Our findings that molecular therapeutics targeted against the tmicroenvironment appear to be more active when administered in series. It is feasible to empirically identify combinations to examine, such as our successful findings with sorafenib and bevacizumab;however, it is possible that there will be interactive agents that may not be intuitively active. We have completed the optimization of relative high throughput assays allowing us to examine a series of endpoints in vitro in 96 well formats. A proof of concept study is starting that will quantitate tumor cell stimulation of endothelial cell tubule network formation in coculture and measure cytokine production. Three of each of ovarian cancer, pancreatic cancer, and melanoma cell lines will be exposed to a pilot of 6 targeted agents spanning from cell surface to downstream signaling targets. A partial least squares analysis will be applied and final models built for validation. Confirmation of success of this pilot project will lead to a more comprehensive examination of agents and shRNA against their primary and potential key targets such that the biochemical cause of activity is identified. These data will be applied to preclinical confirmation and clinical trial development, using the targets validated as proof of concept biomarkers in the trial. Our asparaginase work is nearing completion and the clinical trial is open to accrual with its complementation of biomarker endpoints related to target protein glycosylation and angiogenesis endpoints from the laboratory work. A final set of experiments is evaluating the lack of cell death with short term exposure to asparaginase (ovarian cancer cells) and with longer exposure in the vascular cells. Other cell injury endpoints are under evaluation. We have initiated examination of PARP inhibition in a vascular development model where PARP inhibition, like asparaginase, does not alter viability per se. The behavior and mechanisms are under examination. We have continued our studies of progranulin (pgrn) and secretory leukocyte protease inhibitor (SLPI) in invasion and metastasis of ovarian cancer. Increased aggressiveness and invasive behavior was observed in cells overexpressing SLPI and/or its protease inhibitor mutants. We found that MMP9 expression and protein production are induced by SLPI and protease-null SLPI in ovarian cancer cells. Activation of MMP9 by plasmin cleavage and release is inhibited by SLPI but not protease-null SLPI, attenuating but not blocking the increase in total MMP9 in response to SLPI stimulation. A direct relationship between SLPI and MMP9 quantity (r2=0.9) was observed a tissue microarray of serous but not endometrioid ovarian cancer samples (Gynecologic Oncology Group array). New ongoing work further examines the role of SLPI in invasion and aggressiveness through dissection of component parts of epithelial-mesenchymal transition. SLPI is nontransforming for immortalized 3T3 fibroblasts and HaCaT keratinocytes. However, stable expression of SLPI in HaCaT cells causes upregulation of migration, invasion, and changes of mesenchymal phenotype in the HaCaT cells, with differing degrees based upon the SLPI mutant. These new data combined with the prior survival and paclitaxel-antagonism is the basis for an expected push for internal development of a neutralizing SLPI therapeutic antibody similar to the CR012 used in our paclitaxel study. That clone is no longer available due to intellectual property issues. We will continue our collaboration with Kohn lab alumnus, Prof. R. Alessandro at the Univ. Palermo in their studies, proteomic, biochemical, and biological, of CML. A new direction has been initiated to investigate interactive proteomic signaling between the endothelium and tumor cells in an in vitro model. Success with this model and signal characterization will lead to detailed dissection and in vivo modeling. New work in our side of the collaboration is characterizing the pro-angiogenic activity of exosomes produced by the CML cells as a partial explanation to the pro-angiogenic activity found in the bone marrow of CML patients. Exosome production, trafficking, and subcellular organization are being studied. These latter behaviors will also be examined in our solid tumor ovarian cancer program.